The efforts of the molecular Mechanisms of Tumor Promotion Section are directed at understanding the early events in the interaction of phorbol ester tumor promoters with cells and tissues. Particular attention is being devoted to the analysis of the major phorbol ester receptor, protein kinase C. Novel protocols have been developed for its purification that are more efficient and afford higher yields than is possible with current methods. Purification of monoclonal and polyclonal antibodies to the receptor is in progress. The role of lipids in reconstitution of the receptor has been characterized in detail. Phospholipids differ in whether or not they can reconstitute, in the amounts required for reconstitution, and in the phorbol ester binding affinities of the resultant complex. Diacylglycerols competitively inhibit phorbol ester binding in vitro, consistent with their being the postulated endogenous phorbol ester analogs. Comparison with the homologous phorbol esters yields differences in affinities of only 20- to 80-fold. As expected from the in vitro assays, treatment of intact cells with phospholipase C to generate diacylglycerol endogenously or the exogenous addition of appropriate diacylglycerols likewise inhibits phorbol ester binding competitively in vivo. The inhibition of binding by diacylglycerols suggested that the phorbol ester receptor could recognize the membrane-dissovled form of the phorbol esters. Analysis of the behavior of a series of highly lipophilic phorbol esters confirmed this prediction. Identification of the enzymatic activity association with the phorbol ester receptor has made it possible to analyze the coupling between binding and subsequent response. Most mouse skin tumor promoters, structurally unrelated to the phorbol esters, did not activate protein kinase C in vitro. Unsaturated fatty acids at high concentrations did activate, however. Multiple phorbol ester receptors have been implicated in the heterogeneity of phorbol ester responses. The binding characteristics of intact, cultured keratinocytes change from homogeneous to heterogeneous as differentiation proceeds.